Primers for diagnosing avellino corneal dystrophy

ABSTRACT

The present invention relates to a real-time PCR primer pair and probe for diagnosing Avellino corneal dystrophy, and more particularly to a real-time PCR primer pair and probe for diagnosing Avellino corneal dystrophy, which can accurately diagnose the presence or absence of a mutation in exon 4 of BIGH3 gene, which is responsible for Avellino corneal dystrophy. The use of the primer pair and probe according to the invention can diagnose Avellino corneal dystrophy in a more rapid and accurate manner than a conventional method that uses a DNA chip or PCR.

TECHNICAL FIELD

The present invention relates to a real-time PCR primer pair and probefor diagnosing Avellino corneal dystrophy, and more particularly to sucha real-time PCR primer pair and probe for diagnosing Avellino cornealdystrophy, which can accurately diagnose the presence or absence of amutation in exon 4 of BIGH3 gene, which is responsible for Avellinocorneal dystrophy.

BACKGROUND ART

Corneal dystrophy is an autosomal dominant hereditary disease, whichbegins with a blurry symptom in the center of cornea and graduallyspreads and thus ends up vision loss as a patient gets older. Itincludes Avellino corneal dystrophy, Granular corneal dystrophy, latticetype I corneal dystrophy, Reis-bucklers corneal dystrophy, etc., and iscaused by mutation of a gene coding βIG-H3 protein.

Heterozygous patients suffering from Avellino corneal dystrophy appearto have severe loss of vision as getting older and homozygous patientsappear to have complete loss of vision since 6 years old. Avellinocorneal dystrophy is a newly named disease in 1988, divided fromgenerally called Granular corneal dystrophy because it was found to havediscrete symptoms and genetic foundation. Also, it has been known to bethe most common corneal dystrophy worldwide, 1/340 to 1/1000 ofprevalence rate in Korea (the case of heterozygote) based on geneticanalysis indicates that it is a common dystrophy (Holland, E. J. et al.,Ophthalmology, 99:1564, 1992; Kennedy, S. M. et al., Br. J. Ophthalmol.,80:489, 1996; Dolmetsch, A. M. et al., Can. J. Ophthalmol., 31:29, 1996;Afshari, N. A. et al., Arch. Ophthalmol., 119:16, 2001; Stewart, H. S.Hum. Mutat., 14:126, 1999).

The present inventors has found that if a patient suffering fromheterozygous Avellino corneal dystrophy has LASIK surgery, 2 yearslater, opacity of cornea starts to develop aggressively and eventuallyresults in vision loss (Jun, R. M. et al., Opthalmology, 111:463, 2004).Previously, eye surgery has been performed with an expectation thatLASIK or Excimer Laser surgery would get rid of vision blurriness of apatient suffering from corneal dystrophy. Also, even in Korea,approximately 3 hundred thousand cases of LASIK surgery have beenperformed, which leads to the assumption that 300 people lost theirvision, based on 1/1000 of minimum estimation of heterozygous patientssuffering from Avellino corneal dystrophy. Patients who have undergoneLASIK surgery are mainly in their 20's and 30's carrying out productiveactivities; therefore, their vision loss causes serious troubles in bothsociety and economics.

In addition, after approval of LASIK surgery in year 2000 in USA,African American patients suffering from Avellino corneal dystrophy whounderwent LASIK surgery have been found to lose eye sight, which infersthat plenty of similar cases might be occurring throughout the world.

Therefore, although accurate diagnosis of Avellino corneal dystrophy isrequired to prevent the progression of Avellino corneal dystrophy byLASIK surgery, the diagnosis of Avellino corneal dystrophy is justconducted by microscopic observation of corneal opacity and thus oftendoctors miss latent symptoms of patients to perform LASIK surgery, whichresults in vision loss. Therefore, rapid and precise diagnosis ofcorneal dystrophy is desperately in need.

A DNA chip for detecting a mutation in BIGH3 gene, which is responsiblefor Avellino corneal dystrophy, was developed (Korean Patent Laid-OpenPublication No. 10-2007-0076532). However, the diagnosis of Avellinocorneal dystrophy using said DNA chip disadvantageously require severalsteps, including a step of amplifying DNA in a sample, a step ofhybridizing the amplified DNA with the DNA chip, a step of washing thehybridized DNA chip, and a step of detecting a positive response.

Accordingly, the present inventors have made extensive efforts todevelop a method capable of more efficiently diagnosing Avellino cornealdystrophy, and as a result, have found that, if the diagnosis ofAvellino corneal dystrophy is performed using a pair of primers havingnucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and probes havingnucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14 by a real-timePCR method, Avellino corneal dystrophy can be diagnosed in a more rapidand accurate manner than a conventional method, thereby completing thepresent invention.

DISCLOSURE OF INVENTION

A main object of the present invention is to provide a primer pair andprobe for more efficiently and accurately diagnosing Avellino cornealdystrophy using a real-time PCR method.

To achieve the above object, the present invention provides a real-timePCR primer pair for diagnosing Avellino corneal dystrophy, which isrepresented by nucleotide sequences selected from the group consistingof SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ IDNOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs:13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19and 20, SEQ ID NOs: 21 and 22, and SEQ ID NOs: 23 and 24.

The present invention also provides a real-time PCR probe for diagnosingAvellino corneal dystrophy, which is represented by a nucleotidesequence selected from the group consisting of SEQ ID NO: 25 to SEQ IDNO: 42.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results obtained from the design of real-time PCRprimers and probes. In FIG. 1, “A” shows the results of real-time PCRcarried out using optimal primers and probes, “B” and “C” show theresults of real-time PCR carried out using primers different from thosein “A”.

FIG. 2 shows the results of real-time PCR carried out using real-timePCR primers according to the present invention in order to detect a genemutation causing Avellino corneal dystrophy.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention is directed to a real-time PCRprimer pair for diagnosing Avellino corneal dystrophy, which isrepresented by nucleotide sequences selected from the group consistingof SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ IDNOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs:13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19and 20, SEQ ID NOs: 21 and 22, and SEQ ID NOs: 23 and 24.

Avellino corneal dystrophy is a disease caused by genetic abnormality inwhich the sequence CGC in exon 4 of BIGH3 gene is mutated to CAC so thatarginine at residue of BIGH3 protein is mutated to histidine (R124H).

When a real-time PCR method is carried out using primers of the presentinvention, Avellino corneal dystrophy can be diagnosed in a more rapidand accurate manner than a conventional method that uses a DNA chip.

In the real-time PCR method, it is very difficult to establishtemperature conditions, because experiments using primers and probesshould be carried out in the same temperature conditions. Particularly,if only one mutation position like Avellino corneal dystrophy is to bedetected, primers and probes should be used in temperature conditions inwhich they can bind. Also, the probes can bind in a very limitedtemperature range from 1° C. and 3° C. in a state in which only onenucleotide differs between the probe for detecting a normal gene and theprobe for detecting a mutant gene.

Due to such conditions, it is important to find the same temperatureconditions in which probes and primers can bind to a desired gene.Particularly, it is important to design a mutant probe and a normalprobe such that the temperatures thereof can differ as much as possiblewithin a limited range. In other words, the temperatures for primers andprobes should be well consistent with each other, the temperatureconditions for the forward primer and the reverse primer should be wellconsistent with each other, and the difference between the temperaturesfor the mutant probe and the normal probe should be able to bemaximized. FIG. 1A shows the results of using well-designed primers andprobes, and FIGS. 1B and 1C shows the results of using primers differentfrom those used in FIG. 1A while using the same probes as those used inFIG. 1A. As can be seen therein, the design of the primers and theprobes has a significant effect on reading.

In the present invention, in order to construct optimal primers fordiagnosing Avellino corneal dystrophy using a real-time PCR method,pairs of primers of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ IDNOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22 and SEQ ID NOs: 23 and24 were designed, and real-time PCR was performed using each of thedesigned primer pairs. As a result, it was found that the use of thepair of primers of SEQ ID NOs: 1 and 2 showed the optimal results.

In another aspect, the present invention is directed to a real-time PCRprobe for diagnosing Avellino corneal dystrophy, which is represented bya nucleotide sequence selected from the group consisting of SEQ ID NO:25 to SEQ ID NO: 42.

In the present invention, in order to construct optimal probes fordiagnosing Avellino corneal dystrophy using a real-time PCR method,probes of SEQ ID NOs: 25 to 42 were designed, and real-time PCR wasperformed each of the designed primers. As a result, it was found thatthe use of the probes of SEQ ID NOs: 13 and 14 showed the optimalresults.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are illustrative purposesonly and are not to be construed to limit the scope of the presentinvention. That is, the following steps will be described as oneillustrative ones and do not limit the scope of the present invention.

Example 1 Construction of Real-Time PCR Primers and MGB Probes

In order to construct primers capable of amplifying a region comprisinga mutation in exon 4 of BIGH3 gene, pairs of primers of SEQ ID NOs: 1and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8,SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ IDNOs: 21 and 22 and SEQ ID NOs: 23 and 24 were designed using PrimerExpress 3.0 software (Applied Biosystems U.S.A).

ACD Fw primer: (SEQ ID NO: 1) 5′-TCC ACC ACC ACT CAG CTG TAACD Re primer: (SEQ ID NO: 2) 5′-CCA TCT CAG GCC TCA GCT T (60 bp)AV Fw primer: (SEQ ID NO: 3) 5′-TGC AGC CCT ACC ACT CTC AA AV Re primer:(SEQ ID NO: 4) 5′-AGG CCT CGT TGC TAG G (150 bp) Real Fw primer:(SEQ ID NO: 5) 5′-TAG TCT CTT ATT CTA ATA GA Real Re primer:(SEQ ID NO: 6) 5′-GCT GCA GAC TCT GTG TTT AA (860 bp) ACD Fw2 primer:(SEQ ID NO: 7) 5′-CCA TCC CTC CTT CTG TCT TCT G ACD Re2 primer:(SEQ ID NO: 8) 5′-CGG GCC CCT CCA TCT C (140 bp) ACD Fw3 primer:(SEQ ID NO: 9) 5′-CAG AGA AGG GAG GGT GTG GTT ACD Re3 primer:(SEQ ID NO: 10) 5′-GGG CGA AGA TGG TGA AGC T (190 bp) ACD Fw4 primer:(SEQ ID NO: 11) 5′-TCC TCG TCC TCT CCA CCT GTA ACD Re4 primer:(SEQ ID NO: 12) 5′-AGC TGG CAA GGA GGC CC ACD Fw5 primer:(SEQ ID NO: 13) 5′-TTT GGG CTT TCC CAC ATG C ACD Re5 primer:(SEQ ID NO: 14) 5′-GGC AGA CGG AGG TCA TCT CA ACD Fw6 primer:(SEQ ID NO: 15) 5′-GTA GTA CCG TGC TCT CTG ACD Re6 primer:SEQ ID NO: 16) 5′-AGT TCC CCA TAA GAA TCC CCC ACD Fw7 primer:(SEQ ID NO: 17) 5′-GGC TGG ACC CCC AGA GG ACD Re7 primer:(SEQ ID NO: 18) 5′-ACC CCT CGG GGA AGT AAG G ACD Fw8 primer:(SEQ ID NO: 19) 5′-AAC CTT TAC GAG ACC CTG GGA ACD Re8 primer:(SEQ ID NO: 20) 5′-GAC TCC CAT CCA TCA TGC CC ACD Fw9 primer:(SEQ ID NO: 21) 5′-AGT CGT TGG ATC CAC CAC CA ACD Re9 primer:(SEQ ID NO: 22) 5′-GAC GTC ATT TCC TAC TGT TTC AGG ACD Fw10 primer:(SEQ ID NO: 23) 5′-CCC CCC AGA AAC AGC CTG ACD Re10 primer:(SEQ ID NO: 24) 5′-TTC TAA GGG GTT AAG GAG AAA GCT T

In order to detect a guanine-to-adenine mutation in exon 4 of BIGH3gene, probes of SEQ ID NOs: 25 to 42 were constructed.

The probe binding to a normal gene fragment having no mutation waslabeled with VIC, and the probe binding to a gene fragment having amutation was labeled with FAM, and a minor groove binder (MGB) wasattached to the probe so as to facilitate binding to a complementarygene fragment.

Normal probe 1: (SEQ ID NO: 25) VIC-CAC GGA CCG CAC GGA-NFQ (15 bp)Mutant probe 1: (SEQ ID NO: 26) FAM-CAC GGA CCA CAC GGA-NFQNormal probe 2: (SEQ ID NO: 27) VIC-ACA CGG ACC GCA CG-NFQMutant probe 2: (SEQ ID NO: 28) FAM-ACA CGG ACC ACA CG-NFQ (14 bp)Normal probe 3: (SEQ ID NO: 29) VIC-TAC ACG GAC CGC A-NFQMutant probe 3: (SEQ ID NO: 30) FAM-TAC ACG GAC CAC A-NFQ (13 bp)Normal probe 4: (SEQ ID NO: 31) VIC-CTG TAC ACG GAC CGC ACG-NFQMutant probe 4: (SEQ ID NO: 32) FAM-CTG TAC ACG GAC CAC ACG-NFQ (18 bp)Normal probe 5: (SEQ ID NO: 33) VIC-CTG TAC ACG GAC CGC ACG GAG-NFQMutant probe 5: (SEQ ID NO: 34) FAM-CTG TAC ACG GAC CAC ACG GAG-NFQ(21 bp) Normal probe 6: (SEQ ID NO: 35)VIC-GCT GTA CAC GGA CCG CAC GGA GAA-NFQ Mutant probe 6: (SEQ ID NO: 36)FAM-GCT GTA CAC GGA CCA CAC GGA GAA-NFQ Normal probe 7: (SEQ ID NO: 37)VIC-ACC GCA CGG AGA AGC-NFQ Mutant probe 7: (SEQ ID NO: 38)FAM-ACC ACA CGG AGA AGC-NFQ Normal probe 8: (SEQ ID NO: 39)VIC-ACC GCA CGG AGA AGC TGA GGC-NFQ Mutant probe 8: (SEQ ID NO: 40)FAM-ACC ACA CGG AGA AGC TGA GGC-NFQ Normal probe 8: (SEQ ID NO: 41)VIC-ACC GCA CGG AGA AGC TGA GGC CTG-NFQ Mutant probe 8: (SEQ ID NO: 42)FAM-ACC ACA CGG AGA AGC TGA GGC CTG-NFQ

Example 2 Diagnosis of Avellino Corneal Dystrophy Using Real-Time PCR

Samples were taken from the blood, hair root and oral epithelial cellsof test subjects, and DNA was isolated from the samples. The isolationand purification of DNA were performed using a partial modification ofthe phenol/chloroform extraction method (Miller, SA et al., Nucl. AcidsRes. 16:1215, 1988), and the isolated DNA was dissolved in a suitableamount of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH7.4) and confirmed byelectrophoresis on 1% agarose gel and used as template DNA in PCR.

The PCR reactions were performed using the primers (SEQ ID NOs: 1 to 12)for amplifying the fragment containing the mutation region, and theprobes (SEQ ID NOs: 13 to 24), constructed in Example 1.

25 μg of a master mix containing 10 pmol of each primer and 5 pmol ofeach probe was prepared and used in the PCR reaction.

The real-time PCR reaction was performed in the following conditions: 36cycles each consisting of 10 min at 95° C., 15 sec at 92° C. and 1 minat 60° C., followed by a reaction for 5 min at 60° C.

After each cycle, fluorescence was measured. The sample positive to theVIC dye was diagnosed as having the normal gene, and the sample positiveto the FAM gene was diagnosed as having the mutant gene.

As a result, it could be seen that the use of the primer pair of SEQ IDNOs: 1 and 2 and the probes of SEQ ID NOs: 25 and 26 showed the mostaccurate and effective results (FIG. 2).

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

INDUSTRIAL APPLICABILITY

The use of the primer pair and probe according to the present inventioncan diagnose Avellino corneal dystrophy in a more rapid and accuratemanner than a conventional method that uses a DNA chip or PCR.

1. A real-time PCR primer pair for diagnosing Avellino cornealdystrophy, which is represented by nucleotide sequences selected fromthe group consisting of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ IDNOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, and SEQ ID NOs: 23 and24.
 2. A real-time PCR probe for diagnosing Avellino corneal dystrophy,which is represented by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 25 to SEQ ID NO:
 42. 3. The real-time PCR probefor diagnosing Avellino corneal dystrophy of claim 2, wherein thereal-time PCR probe is labeled with VIC or FAM.